The reference numbers used in this section and throughout this disclosure refer to the documents set forth in the “References” section herein.
DNA polymerase activity is indispensable for genome replication and organism propagation across all biological domains (1-3). Since its initial characterization (4), the ability to harness DNA polymerase activity in vitro has become a fundamental tool in the field of molecular biology research (5). Above and beyond its established importance in research, in vitro measurement of DNA polymerase activity potentially offers numerous useful applications within the pharmaceutical and clinical setting. For instance, since bacterial DNA polymerase is actively being targeted for development of novel antimicrobial agents (6, 7), a rapid and sensitive assay capable of measuring DNA polymerase activity is desirable. Also, loss or gain of DNA polymerase activity is intimately involved in human disease. For example, emerging links between DNA polymerase activity and genetic aberrations are designating the enzyme as a target for anti-cancer therapies (8, 9). Deficiencies in DNA polymerase activity have also been linked to mitochondrial disorders (10). Furthermore, measurement of DNA polymerase activity has the potential to be used as a rapid and sensitive diagnostic tool, capable of detecting virtually any organism harboring active DNA polymerase within a given environmental or biological matrix where sterility is expected.
The most common method used to measure DNA polymerase activity in vitro depends upon incorporation of radiolabeled nucleotides (11). However, routine use of such DNA polymerase assays is undesirable due to the inherent risks and restrictions associated with radioisotopes. Consequently, over the past few decades numerous non-radioactive in vitro polymerase assays have been developed. Some rely upon the measurement of fluorescence generated by DNA polymerase-mediated release of single stranded binding protein (12) or binding of PICOGREEN dsDNA reagent to double stranded DNA (13,14). Other methods rely on microplate coupling and detection of fluorescently-labeled nucleotides (15). More recently, molecular beacon-based (16) and electrochemical-based (17) DNA polymerase assays have been developed. Despite successfully averting the use of radioactivity, the above assays are limited by such factors as poor sensitivity, a small linear dynamic range of measurement, or the use of purified polymerase.
As will be apparent to those skilled in the relevant art, the measurement of DNA polymerase extension activity in accordance with the present invention as described herein represents a useful tool with far reaching applications such as, but not limited to, screening candidate-polymerase inhibitors in vitro, or detecting the presence any microbe (harboring active DNA polymerases) within a diverse range of sample types. This is a substantial improvement over the state of the present art, because if intended for these purposes, routine use of traditional polymerase assays that incorporate radiolabeled nucleotides is unattractive. Consequently, numerous non-radioactive DNA polymerase extension assays have been developed in recent decades. Despite successfully averting the use of radioactivity, current fluorescence-based DNA polymerase assays also suffer from various deficiencies. For example, detection of DNA polymerase activity via several existing non-radioactive assays is dependent upon the binding of PICOGREEN dsDNA reagent to newly-generated double stranded DNA (13,14). If intended to analyze DNA polymerase activity from freshly lysed organisms, PICOGREEN.-based assays would likely be hampered by background fluorescence via binding of PICOGREEN dsDNA reagent to genomic DNA. Microplate-based DNA polymerase assays have also been developed (15). Decreased sensitivity of microplate-based assays can be expected for numerous reasons, including dependence upon intermediate binding of either product or substrate to a microplate and/or inefficient incorporation of modified dNTPs by DNA polymerase. More recently, real-time measurement of DNA polymerase activity via molecular beacons has been described (16). Despite improved sensitivity, direct measurement of molecular beacon fluorescence could also potentially be hindered by exposure to crude cellular lysates.